Angiotensin II Inhibits Insulin Receptor Signaling in Adipose Cells.

Angiotensin II (Ang II) is a critical regulator of insulin signaling in the cardiovascular system and metabolic tissues. However, in adipose cells, the regulatory role of Ang II on insulin actions remains to be elucidated. The effect of Ang II on insulin-induced insulin receptor (IR) phosphorylation, Akt activation, and glucose uptake was examined in 3T3-L1 adipocytes. In these cells, Ang II specifically inhibited insulin-stimulated IR and insulin receptor substrate-1 (IRS-1) tyrosine-phosphorylation, Akt activation, and glucose uptake in a time-dependent manner. These inhibitory actions were associated with increased phosphorylation of the IR at serine residues. Interestingly, Ang II-induced serine-phosphorylation of IRS was not detected, suggesting that Ang II-induced desensitization begins from IR regulation itself. PKC inhibition by BIM I restored the inhibitory effect of Ang II on insulin actions. We also found that Ang II promoted activation of several PKC isoforms, including PKCα/ßI/ßII/δ, and its association with the IR, particularly PKCßII, showed the highest interaction. Finally, we also found a similar regulatory effect of Ang II in isolated adipocytes, where insulin-induced Akt phosphorylation was inhibited by Ang II, an effect that was prevented by PKC inhibitors. These results suggest that Ang II may lead to insulin resistance through PKC activation in adipocytes.

Angiotensin II induces vascular endocannabinoid release, which attenuates its vasoconstrictor effect via CB1 cannabinoid receptors.

In the vascular system angiotensin II (Ang II) causes vasoconstriction via the activation of type 1 angiotensin receptors. Earlier reports have shown that in cellular expression systems diacylglycerol produced during type 1 angiotensin receptor signaling can be converted to 2-arachidonoylglycerol, an important endocannabinoid. Because activation of CB(1) cannabinoid receptors (CB(1)R) induces vasodilation and reduces blood pressure, we have tested the hypothesis that Ang II-induced 2-arachidonoylglycerol release can modulate its vasoconstrictor action in vascular tissue. Rat and mouse skeletal muscle arterioles and mouse saphenous arteries were isolated, pressurized, and subjected to microangiometry. Vascular expression of CB(1)R was demonstrated using Western blot and RT-PCR. In accordance with the functional relevance of these receptors WIN55212, a CB(1)R agonist, caused vasodilation, which was absent in CB(1)R knock-out mice. Inhibition of CB(1)Rs using O2050, a neutral antagonist, enhanced the vasoconstrictor effect of Ang II in wild type but not in CB(1)R knock-out mice. Inverse agonists of CB(1)R (SR141716 and AM251) and inhibition of diacylglycerol lipase using tetrahydrolipstatin also augmented the Ang II-induced vasoconstriction, suggesting that endocannabinoid release modulates this process via CB(1)R activation. This effect was independent of nitric-oxide synthase activity and endothelial function. These data demonstrate that Ang II stimulates vascular endocannabinoid formation, which attenuates its vasoconstrictor effect, suggesting that endocannabinoid release from the vascular wall and CB(1)R activation reduces the vasoconstrictor and hypertensive effects of Ang II.

The Concise Guide to PHARMACOLOGY 2023/24: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.16177. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

The relationship between pulsatile GnRH secretion and cAMP production in immortalized GnRH neurons.

In perifused immortalized GnRH neurons (GT1-7), simultaneous measurements of GnRH and cAMP revealed that the secretory profiles for both GnRH and cAMP are pulsatile. An analysis of GnRH and cAMP pulses in 16 independent experiments revealed that 25% of pulses coincide. Inversion of the peak and nadir levels was found in 33% and random relationship between GnRH and cAMP found in 42% of analyzed pulses. The random relation between GnRH and cAMP pulse resets to synchronous after an inverse relation between pulses occurred during the major GnRH release, indicating that GnRH acts as a switching mechanism to synchronize cAMP and GnRH release in perifused GT1-7 neurons. Activation of GnRH receptors with increasing agonist concentrations caused a biphasic change in cAMP levels. Low nanomolar concentrations increased cAMP production, but at high concentrations the initial increase was followed by a rapid decline to below the basal level. Blockade of the GnRH receptors by peptide and nonpeptide antagonists generated monotonic nonpulsatile increases in both GnRH and cAMP production. These findings indicate that cAMP positively regulates GnRH secretion but does not participate in the mechanism of pulsatile GnRH release.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Expression and function of ATIP/MTUS1 in human prostate cancer cell lines.

BACKGROUND: We have previously demonstrated Ang II type 2 (AT(2)-) receptor-mediated inhibition of EGF-induced prostate cancer cell growth in androgen-dependent (LNCaP) and independent (PC3) prostate cancer cell lines. METHODS: To explore the signaling pathways involved in this inhibitory effect, we examined the interaction of the AT(2)-receptor with its novel regulatory partner ATIP using real time PCR, over-expression, siRNA and [(3)H]thymidine incorporation assays. RESULTS: The results in human prostate cancer cell lines demonstrate the presence of ATIP in both cell lines examined, and suggest that (i) the AT(2)-receptor through an interaction with ATIP mediates an anti-growth factor effect in both androgen-dependent and androgen-independent cell lines; (ii) ATIP expression decreases as the rate of cell growth and androgen-independence increase; and (iii) EGF may act on cell growth in part by reducing the content of ATIP present in the cells. CONCLUSIONS: The results support our earlier proposal in normal cell lines that ATIP is an important component of the cellular response to AT(2)-receptor activation. The results further suggest that a critical level of ATIP is required to mediate the effect of AT(2)-receptor activation to inhibit EGF mediated increases in cell growth. They also suggest that EGF may in part induce cell growth by suppressing the level of ATIP expression.

Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor.

Agonist-induced endocytosis and processing of the G protein-coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)- or hemagglutinin epitope-tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5-GFP and Rab4-GFP in early endosomes, and subsequently with Rab11-GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand-receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa(R)-transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine-Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase-dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

Converse regulatory functions of estrogen receptor-alpha and -beta subtypes expressed in hypothalamic gonadotropin-releasing hormone neurons.

Estradiol (E(2)) acts as a potent feedback molecule between the ovary and hypothalamic GnRH neurons, and exerts both positive and negative regulatory actions on GnRH synthesis and secretion. However, the extent to which these actions are mediated by estrogen receptors (ERs) expressed in GnRH neurons has been controversial. In this study, Single-cell RT-PCR revealed the expression of both ERalpha and ERbeta isoforms in cultured fetal and adult rat hypothalamic GnRH neurons. Both ERalpha and ERbeta or individual ERs were expressed in 94% of cultured fetal GnRH neurons. In adult female rats at diestrus, 68% of GnRH neurons expressed ERs, followed by 54% in estrus and 19% in proestrus. Expression of individual ERs was found in 24% of adult male GnRH neurons. ERalpha exerted marked G(i)-mediated inhibitory effects on spontaneous action potential (AP) firing, cAMP production, and pulsatile GnRH secretion, indicating its capacity for negative regulation of GnRH neuronal function. In contrast, increased E(2) concentration and ERbeta agonists increase the rate of AP firing, GnRH secretion, and cAMP production, consistent with ERbeta-dependent positive regulation of GnRH secretion. Consonant with the coupling of ERalpha to pertussis toxin-sensitive G(i/o) proteins, E(2) also activates G protein-activated inwardly rectifying potassium channels, decreasing membrane excitability and slowing the firing of spontaneous APs in hypothalamic GnRH neurons. These findings demonstrate that the dual actions of E(2) on GnRH neuronal membrane excitability, cAMP production, and GnRH secretion are mediated by the dose-dependent activation of ERalpha and ERbeta expressed in hypothalamic GnRH neurons.

CRF1 Receptor Signaling via the ERK1/2-MAP and Akt Kinase Cascades: Roles of Src, EGF Receptor, and PI3-Kinase Mechanisms.

In the present study, we determined the cellular regulators of ERK1/2 and Akt signaling pathways in response to human CRF1 receptor (CRF1R) activation in transfected COS-7 cells. We found that Pertussis Toxin (PTX) treatment or sequestering Gßγ reduced CRF1R-mediated activation of ERK1/2, suggesting the involvement of a Gi-linked cascade. Neither Gs/PKA nor Gq/PKC were associated with ERK1/2 activation. Besides, CRF induced EGF receptor (EGFR) phosphorylation at Tyr1068, and selective inhibition of EGFR kinase activity by AG1478 strongly inhibited the CRF1R-mediated phosphorylation of ERK1/2, indicating the participation of EGFR transactivation. Furthermore, CRF-induced ERK1/2 phosphorylation was not altered by pretreatment with batimastat, GM6001, or an HB-EGF antibody indicating that metalloproteinase processing of HB-EGF ligands is not required for the CRF-mediated EGFR transactivation. We also observed that CRF induced Src and PYK2 phosphorylation in a Gßγ-dependent manner. Additionally, using the specific Src kinase inhibitor PP2 and the dominant-negative-SrcYF-KM, it was revealed that CRF-stimulated ERK1/2 phosphorylation depends on Src activation. PP2 also blocked the effect of CRF on Src and EGFR (Tyr845) phosphorylation, further demonstrating the centrality of Src. We identified the formation of a protein complex consisting of CRF1R, Src, and EGFR facilitates EGFR transactivation and CRF1R-mediated signaling. CRF stimulated Akt phosphorylation, which was dependent on Gi/ßγ subunits, and Src activation, however, was only slightly dependent on EGFR transactivation. Moreover, PI3K inhibitors were able to inhibit not only the CRF-induced phosphorylation of Akt, as expected, but also ERK1/2 activation by CRF suggesting a PI3K dependency in the CRF1R ERK signaling. Finally, CRF-stimulated ERK1/2 activation was similar in the wild-type CRF1R and the phosphorylation-deficient CRF1R-Δ386 mutant, which has impaired agonist-dependent ß-arrestin-2 recruitment; however, this situation may have resulted from the low ß-arrestin expression in the COS-7 cells. When ß-arrestin-2 was overexpressed in COS-7 cells, CRF-stimulated ERK1/2 phosphorylation was markedly upregulated. These findings indicate that on the base of a constitutive CRF1R/EGFR interaction, the Gi/ßγ subunits upstream activation of Src, PYK2, PI3K, and transactivation of the EGFR are required for CRF1R signaling via the ERK1/2-MAP kinase pathway. In contrast, Akt activation via CRF1R is mediated by the Src/PI3K pathway with little contribution of EGFR transactivation.

Differential signaling pathways in angiotensin II- and epidermal growth factor-stimulated hepatic C9 cells.

Caveolin1 (Cav1) is an important component of the plasmamembrane microdomains, such as caveolae/lipid rafts, that are associated with angiotensin II type 1 (AT(1)) and epidermal growth factor (EGF) receptors in certain cell types. The interactions of Cav1 with other signaling molecules that mediate AT(1) receptor function were analyzed in angiotensin II (Ang II)- and EGF-stimulated hepatic C9 cells. This study demonstrated that cholesterol-rich domains mediate the actions of early upstream signaling molecules such as Src and intracellular Ca(2+) in cells stimulated by Ang II, but not by EGF, and that Cav1 has a scaffolding role in the process of mitogen-activated protein kinase activation. Furthermore, Cav1 phosphorylation by Ang II and EGF was regulated by intracellular Ca(2+) and Src, further indicating reciprocal interactions among Cav1, Src, and intracellular Ca(2+) through the AT(1) receptor. Phosphorylation of Cav1 and the EGF receptor by Ang II, but not of extracellular signal-regulated kinase 1/2, was dependent on intracellular Ca(2+). The phosphatidylinositol 3-kinase inhibitors, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin, differentially modulated both Cav1 and EGF receptor activation by Ang II through intracellular Ca(2+). These findings further demonstrate the importance of Cav1 in conjunction with the receptor-mediated signaling pathways involved in cell proliferation and survival. It is clear that differential signaling pathways are operative in Ang II- and EGF-stimulated C9 cells and that cholesterol-enriched microdomains are essential components in cellular signaling processes that are dependent on specific agonists and/or cell types.

Expression of a functional g protein-coupled receptor 54-kisspeptin autoregulatory system in hypothalamic gonadotropin-releasing hormone neurons.

The G protein-coupled receptor 54 (GPR54) and its endogenous ligand, kisspeptin, are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons with primers for GnRH, kisspeptin-1, and GPR54 revealed expression of all three gene products. Also, constitutive and GnRH agonist-induced bioluminescence resonance energy transfer between Renilla luciferase-tagged GnRH receptor and GPR54 tagged with green fluorescent protein, expressed in human embryonic kidney 293 cells, revealed heterooligomerization of the two receptors. Whole cell patch-clamp recordings from identified GnRH neurons showed initial depolarizing effects of kisspeptin on membrane potential, followed by increased action potential firing. In perifusion studies, treatment of GT1-7 neuronal cells with kisspeptin-10 increased GnRH peak amplitude and duration. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1-7 cells were detected by a specific RIA and was significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. Stimulation of GnRH secretion by kisspeptin and the opposing effects of GnRH on kisspeptin secretion indicate that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to regulate GnRH and kisspeptin secretion from GnRH neurons.

TACE-dependent EGF receptor activation in angiotensin-II-induced kidney disease.

Angiotensin II (Ang II) has been implicated in the development of cardiovascular disorders and chronic kidney disease (CKD). Ang II causes renal lesions through the activation of tumor necrosis factor (TNF)-alpha-converting enzyme (TACE, also called a disintegrin and a metalloproteinase domain 17) and the release of transforming growth factor (TGF)-alpha, which binds to and activates the epidermal growth factor receptor. Renal lesions such as glomerulosclerosis, tubular atrophy, fibrosis, mononuclear cell infiltration and proteinuria following chronic Ang II infusion are substantially reduced in mice lacking TGF-alpha and those given a specific TACE inhibitor. These findings indicate that the selective inhibition of renal TACE could have therapeutic potential in the treatment of CKD.

Protein phosphatase 5 as a negative key regulator of Raf-1 activation.

Growth factors such as the epidermal growth factor cause sequential activation of receptor tyrosine kinases, adaptor molecules and the Raf-MEK-ERK pathway. The kinetics and intensity of these signals are dependent on the balance between phosphorylation and dephosphorylation of these molecules by numerous kinases and phosphatases, respectively. Recently, protein phosphatase 5 has been characterized as a key dephosphorylation regulator of Raf-1 activation in growth factor-mediated signaling, leading to attenuation of the MEK-ERK cascade.

Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II.

Angiotensin II (Ang II) activates a wide spectrum of signaling responses via the AT1 receptor (AT1R) that mediate its physiological control of blood pressure, thirst, and sodium balance and its diverse pathological actions in cardiovascular, renal, and other cell types. Ang II-induced AT1R activation via Gq/11 stimulates phospholipases A2, C, and D, and activates inositol trisphosphate/Ca2+ signaling, protein kinase C isoforms, and MAPKs, as well as several tyrosine kinases (Pyk2, Src, Tyk2, FAK), scaffold proteins (G protein-coupled receptor kinase-interacting protein 1, p130Cas, paxillin, vinculin), receptor tyrosine kinases, and the nuclear factor-kappaB pathway. The AT1R also signals via Gi/o and G11/12 and stimulates G protein-independent signaling pathways, such as beta-arrestin-mediated MAPK activation and the Jak/STAT. Alterations in homo- or heterodimerization of the AT1R may also contribute to its pathophysiological roles. Many of the deleterious actions of AT1R activation are initiated by locally generated, rather than circulating, Ang II and are concomitant with the harmful effects of aldosterone in the cardiovascular system. AT1R-mediated overproduction of reactive oxygen species has potent growth-promoting, proinflammatory, and profibrotic actions by exerting positive feedback effects that amplify its signaling in cardiovascular cells, leukocytes, and monocytes. In addition to its roles in cardiovascular and renal disease, agonist-induced activation of the AT1R also participates in the development of metabolic diseases and promotes tumor progression and metastasis through its growth-promoting and proangiogenic activities. The recognition of Ang II's pathogenic actions is leading to novel clinical applications of angiotensin-converting enzyme inhibitors and AT1R antagonists, in addition to their established therapeutic actions in essential hypertension.

Serotonin (5-HT) receptor subtypes mediate specific modes of 5-HT-induced signaling and regulation of neurosecretion in gonadotropin-releasing hormone neurons.

Serotonin (5-HT), the endogenous nonselective 5-HT receptor agonist, activates the inositol 1,4,5-triphosphate/calcium (InsP3/Ca2+) signaling pathway and exerts both stimulatory and inhibitory actions on cAMP production and GnRH release in immortalized GnRH neurons. The high degree of similarity between the signaling and secretory responses elicited by GnRH and 5-HT prompted us to target specific 5-HT receptor subtypes to deconvolute the complex actions of these agonists on signal transduction and GnRH release. Specific mRNA transcripts for 5-HT1A, 5-HT2C, 5-HT4, and 5-HT7 were identified in immortalized GnRH neurons (GT1-7). The rate of firing of spontaneous action potentials (APs) by hypothalamic GnRH neurons and cAMP production and pulsatile GnRH release in GT17 cells were profoundly inhibited during activation of the Gi-coupled 5-HT1A receptor. Treatment with a selective agonist to activate the Gq-coupled 5-HT2C receptor increased the rate of firing of spontaneous APs, stimulated InsP3 production and caused a delayed increase in GnRH release. Selective activation of the Gs-coupled 5-HT4 receptor also increased the rate of firing of APs, stimulated cAMP production, and caused a sustained and robust increase in GnRH release. The ability of 5-HT receptor subtypes expressed in GnRH neurons to activate single or multiple G proteins in a time- and dose-dependent manner differentially regulates the phospholipase C/InsP3/Ca2+, and adenylyl cyclase/cAMP signaling pathways, and thereby regulates the frequency and amplitude of pulsatile GnRH release. This process, in conjunction with the modulation of spontaneous electrical activity of the GnRH neuron, contributes to the control of the pulsatile mode of neuropeptide secretion that is characteristic of GnRH neuronal function in vivo and in vitro.

Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells.

G protein-coupled receptors (GPCRs) such as angiotensin II, bradykinin and endothelin-1 (ET-1) are critically involved in the regulation of adrenal function, including aldosterone production from zona glomerulosa cells. Whereas, substantial data are available on the signaling mechanisms of ET-1 in cardiovascular tissues, such information in adrenal glomerulosa cells is lacking. Bovine adrenal glomerulosa (BAG) cells express receptors for endothelin-1 (ET-1) and their stimulation caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), extracellularly regulated signal kinases (ERK1/2), and their dependent proteins, p90 ribosomal S6 kinase (RSK-1) and CREB. ET-1 elicited these responses predominantly through activation of a G(i)-linked cascade with a minor contribution from the G(q)/PKC pathway. Whereas, selective inhibition of EGF-R kinase with AG1478 caused complete inhibition of EGF-induced ERK/RSK-1/CREB activation, it caused only partial reduction (30-40%) of such ET-1-induced responses. Consistent with this, inhibition of matrix metalloproteinases (MMPs) with GM6001 reduced ERK1/2 activation by ET-1, consistent with partial involvement of the MMP-dependent EGF-R activation in this cascade. Activation of ERK/RSK-1/CREB by both ET-1 and EGF was abolished by inhibition of Src, indicating its central role in ET-1 signaling in BAG cells. Moreover, the signaling characteristics of ET-1 in cultured BAG cells closely resembled those observed in clonal adrenocortical H295R cells. The ET-1-induced proliferation of BAG and H295 R cells was much smaller than that induced by Ang II or FGF. These data demonstrate that ET-1 causes ERK/RSK-1/CREB phosphorylation predominantly through activation of G(i) and Src, with a minor contribution from MMP-dependent EGF-R transactivation.

Roles of LPA3 and COX-2 in implantation.

Lysophosphatidic acid (LPA) is a lipid-derived G-protein-coupled receptor (GPCR) agonist that is involved in a variety of physiological and pathological processes, including cell survival, proliferation and differentiation, cytoskeletal rearrangement, cell-cell interactions, tumorigenesis and cell invasion. LPA also stimulates oocyte maturation, the preimplantation development of two- or four-cell embryos to the blastocyst stage and embryo transport in the oviduct. Recent studies revealed that targeted deletion of the LPA(3) receptor results in delayed implantation and altered embryo spacing, and significantly reduced litter size in mice. This was attributable to selective downregulation of uterine cyclooxygenase-2 (COX-2), which generates prostaglandins (PGs) E(2) and I(2). Exogenous administration of PGE(2) or the PGI(2) analogue, carba-prostacyclin, to LPA(3)-deficient female mice rescued delayed implantation but did not prevent defects in embryo spacing. These findings indicate that LPA-induced COX-2 induction has a crucial role in implantation and mammalian reproduction.

Mechanisms of extracellularly regulated kinases 1/2 activation in adrenal glomerulosa cells by lysophosphatidic acid and epidermal growth factor.

The regulation of adrenal function, including aldosterone production from adrenal glomerulosa cells, is dependent on a variety of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In many cell types, GPCR-mediated MAPK activation is mediated through transactivation of RTKs, in particular the epidermal growth factor (EGF) receptor (EGF-R). However, the extent to which this cross-communication between GPCRs and RTKs is operative in the adrenal glomerulosa has not been defined. Bovine adrenal glomerulosa cells express receptors for lysophosphatidic acid (LPA) and EGF. In cultured bovine adrenal glomerulosa cells, LPA, which is predominantly coupled to Gi and partially to Gq/protein kinase C alpha and epsilon, caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), EGF-R, protein kinase B/Akt, extracellularly regulated signal kinases 1/2, and their dependent protein, p90 ribosomal S6 kinase. Overexpression of dominant negative mutants of Ras or EGF-R, and selective inhibition of EGF-R kinase with AG1478, significantly reduced LPA-induced ERK1/2 phosphorylation. However, this was not impaired by inhibition of matrix metalloproteinase (MMP) and heparin-binding EGF. LPA-induced ERK1/2 activation occurs predominantly through EGF-R transactivation by Gi/Src and partly through activation of protein kinase C, which acts downstream of EGF-R and Ras. In contrast, LPA-induced phosphorylation of Shc and ERK1/2 in clonal hepatocytes (C9 cells) was primarily mediated through MMP-dependent transactivation of the EGF-R. These observations in adrenal glomerulosa and hepatic cells demonstrate that LPA phosphorylates ERK1/2 through EGF-R transactivation in a MMP-dependent or -independent manner in individual target cells. This reflects the ability of GPCRs expressed in cell lines and neoplastic cells to utilize distinct signaling pathways that can elicit altered responses compared with those of native tissues.

A central role of EGF receptor transactivation in angiotensin II -induced cardiac hypertrophy.

In addition to their physiological roles in the cardiovascular system (CVS), G-protein-coupled receptor (GPCR) agonists such as noradrenaline, endothelin-1 and angiotensin II (Ang II) are known to be involved in the development of cardiac hypertrophy. Recent studies using targeted overexpression of the angiotensin AT(1) receptor in cardiomyocytes suggest that Ang II can directly promote the growth of cardiomyocytes via transactivation of the epidermal growth factor (EGF) receptor and subsequent activation of mitogen-activated protein kinases (MAPKs). This process is mediated by the production of heparin-binding EGF (HB-EGF) by metalloproteases. Blockade of the generation of HB-EGF by metalloprotease inhibitors, or abrogation of EGF receptor kinase activity by selective pharmacological inhibitors or antisense oligonucleotides, protects against Ang II-mediated cardiac hypertrophy. These approaches offer a potential therapeutic strategy to prevent cardiac remodeling and hypertrophy, and possibly prevent progression to heart failure.

Matrix metalloproteinase-dependent EGF receptor activation in hypertension and left ventricular hypertrophy.

Agonist stimulation of certain G protein-coupled receptors (GPCRs) causes shedding of heparin-binding epidermal growth factor (HB-EGF) through activation of matrix metalloproteinases (MMPs), with subsequent transactivation of the EGF receptor. MMPs are widely expressed, and their dysregulated expression is crucial in cancer, inflammation, and cardiovascular remodeling. Recent studies in hypertensive animals have shown enhanced expression and activation of MMPs and EGF receptors, and their inhibition attenuates cardiac hypertrophy, vasoconstriction and hypertension induced by GPCR agonists such as angiotensin II, endothelin-1 and phenylepherine. These findings suggest that selective inhibition of MMPs might have therapeutic potential in hypertension and other cardiovascular diseases.